Purpose: Response rates to immune checkpoint blockade (ICB; anti-PD-1/anti-CTLA-4) correlate with the extent of tumor immune infiltrate, but the mechanisms underlying the recruitment of T cells following therapy are poorly characterized. A greater understanding of these processes may see the development of therapeutic interventions that enhance T-cell recruitment and, consequently, improved patient outcomes. We therefore investigated the chemokines essential for immune cell recruitment and subsequent therapeutic efficacy of these immunotherapies. Experimental Design: The chemokines upregulated by dual PD-1/CTLA-4 blockade were assessed using NanoStringbased analysis with results confirmed at the protein level by flow cytometry and cytometric bead array. Blocking/neutralizing antibodies confirmed the requirement for key chemokines/cytokines and immune effector cells. Results were confirmed in patients treated with immune checkpoint inhibitors using single-cell RNA-sequencing (RNA-seq) and paired survival analyses. Results: The CXCR3 ligands, CXCL9 and CXCL10, were significantly upregulated following dual PD-1/CTLA-4 blockade and both CD8 þ T-cell infiltration and therapeutic efficacy were CXCR3 dependent. In both murine models and patients undergoing immunotherapy, macrophages were the predominant source of CXCL9 and their depletion abrogated CD8 þ T-cell infiltration and the therapeutic efficacy of dual ICB. Single-cell RNA-seq analysis of patient tumor-infiltrating lymphocytes (TIL) revealed that CXCL9/ 10/11 was predominantly expressed by macrophages following ICB and we identified a distinct macrophage signature that was associated with positive responses to ICB. Conclusions: These data underline the fundamental importance of macrophage-derived CXCR3 ligands for the therapeutic efficacy of ICB and highlight the potential of manipulating this axis to enhance patient responses.

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Adenosine is an immunosuppressive factor that limits anti-tumor immunity through the suppression of multiple immune subsets including T cells via activation of the adenosine A2A receptor (A2AR). Using both murine and human chimeric antigen receptor (CAR) T cells, here we show that targeting A2AR with a clinically relevant CRISPR/Cas9 strategy significantly enhances their in vivo efficacy, leading to improved survival of mice. Effects evoked by CRISPR/Cas9 mediated gene deletion of A2AR are superior to shRNA mediated knockdown or pharmacological blockade of A2AR. Mechanistically, human A2AR-edited CAR T cells are significantly resistant to adenosine-mediated transcriptional changes, resulting in enhanced production of cytokines including IFNγ and TNF, and increased expression of JAK-STAT signaling pathway associated genes. A2AR deficient CAR T cells are well tolerated and do not induce overt pathologies in mice, supporting the use of CRISPR/Cas9 to target A2AR for the improvement of CAR T cell function in the clinic.

Immunotherapy is widely accepted as a powerful new treatment modality for the treatment of cancer. The most successful form of immunotherapy to date has been the blockade of the immune checkpoints PD-1 and CTLA-4. Combining inhibitors of both PD-1 and CTLA-4 increases the proportion of patients who respond to immunotherapy. However, most patients still do not respond to checkpoint inhibitors, and prognostic biomarkers are currently lacking. Therefore, a better understanding of the mechanism by which these checkpoint inhibitors enhance antitumor immune responses is required to more accurately predict which patients are likely to respond and further enhance this treatment modality. Our current study of two mouse tumor models revealed that CD4þ

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The function of MR1-restricted mucosal-associated invariant T (MAIT) cells in tumor immunity is unclear. Here we show that MAIT cell-deficient mice have enhanced NK cell-dependent control of metastatic B16F10 tumor growth relative to control mice. Analyses of this interplay in human tumor samples reveal that high expression of a MAIT cell gene signature negatively impacts the prognostic significance of NK cells. Paradoxically, pre-pulsing tumors with MAIT cell antigens, or activating MAIT cells in vivo, enhances anti-tumor immunity in B16F10 and E0771 mouse tumor models, including in the context of established metastasis. These effects are associated with enhanced NK cell responses and increased expression of both IFN-γ-dependent and inflammatory genes in NK cells. Importantly, activated human MAIT cells also promote the function of NK cells isolated from patient tumor samples. Our results thus describe an activation-dependent, MAIT cell-mediated regulation of NK cells, and suggest a potential therapeutic avenue for cancer treatment.

MR1-restricted mucosal-associated invariant T (MAIT) cells recognize microbial metabolites and play an important role in immunity to infection, however, the role they play in tumor immunity is unclear. Here we show that MAIT cell-deficient mice are more resistant to subcutaneous and lung metastasis B16F10 tumor growth compared to control mice, an effect that was associated with enhanced NK cell numbers and was NK cell-dependent. Analysis of this interplay in cancer patients also revealed that a high expression of a novel MAIT gene signature negatively impacted the prognostic significance of NK cells. Paradoxically, pre-pulsing tumors with MAIT cell antigens, or antigen-mediated MAIT cell activation in vivo, enhanced immunity against B16F10 and E0771 lung tumor metastasis. Furthermore, MAIT cell activation effectively reduced metastatic burden in a more stringent model of established lung metastases in mice. These effects were associated with enhanced NK cell responses and increased expression of both IFNγ-dependent and inflammatory genes in NK cells, which was neutralized by IFNγ blockade. Importantly, activated human MAIT cells also enhanced the function of NK cells isolated from patient tumor samples. These findings provide insight into the contrasting roles that MAIT cells can play in controlling anti-tumor immune responses depending on their activation status, in both mice and humans, and suggest potential therapeutic avenues for exploiting their potential anti-tumor properties for cancer treatment.

Multivalent structures can provide multiple interactions at a target site and improve binding affinity. The multivalent presentation of the anti‐tumour heptapeptide, SNTSESF, was investigated. This peptide's activity has been attributed to blockade of the PD‐1 receptor‐mediated signalling pathway. Two and four peptide units were conjugated to poly ethoxy ethyl glycinamide (PEE−G) scaffolds to prepare high‐purity products. These conjugates and the peptide were examined in a mouse model implanted with GL261 tumours that indicated that presenting more than two copies of peptide SNTSESF on the dendritic scaffold does not increase anti‐tumour activity per peptide. The fluorescent labelled peptide and most active multivalent peptide conjugate were therefore screened for their interaction with the human PD−L1 protein in a fluorescence polarisation assay. No indication of a specific SNTSESF peptide/PD−L1 interaction was observed. This finding was further supported by a molecular modelling binding study.